The effect of nitric oxide on vaccinia virus-encoded ribonucleotide reductase.
Growth inhibition of the DNA virus vaccinia (VACV) by NO is known to occur at the level of DNA synthesis. This inhibition is partially reversed by addition of deoxyribonucleosides, suggesting that NO or NO-related species inhibit viral ribonucleotide reductase (RR). However, the effect of NO on VACV-encoded RR or other DNA-synthesizing enzymes has not been demonstrated. In order to study the effects of NO on VACV-encoded RR, DNA polymerase (DNA pol) and thymidine kinase (TK), we generated a VACV recombinant expressing murine macrophage iNOS under control of a VACV early/late promoter p7.5. Using this recombinant, we demonstrate that expression of iNOS and the resulting production of NO inhibit activity of the viral RR, but not of viral DNA pol and TK. This NO-mediated inhibition of viral RR occurred around the same time as the increase of ADP levels, while it preceded the block in VACV DNA synthesis and the decrease of ATP levels. In addition, we tested the effects of DPTA/NONOate on the growth of different VACV mutants. Fold-inhibition of the growth of VACV deletion mutant for TK was comparable to that of wild-type VACV. VACV containing amplification of the gene for the small subunit of RR appeared to be least sensitive to DPTA/NONOate, while VACV deletion mutant for the large subunit of RR was most sensitive. The results provide a direct evidence for NO-mediated inhibition of VACV-encoded RR.
Fujikura
Yuzo
Y
Institute of Pathological Physiology, 1st Medical Faculty, Charles University, Prague, Czech Republic.
Kudlackova
Pavla
P
Vokurka
Martin
M
Krijt
Jakub
J
Melkova
Zora
Z
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000475:Alkenes / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D051379:Mice; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013937:Thymidine Kinase / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000475:Alkenes; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C453079:dipropylenetriamine-NONOate; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D012264:Ribonucleotide Reductases; D013937:Thymidine Kinase; D004259:DNA-Directed DNA Polymerase